Targeted delivery to human diseases and disorders

ABSTRACT

The present invention provides a system presenting site-specific accumulation through a ligand that specifically targets a receptor overexpressed on the surface of specific cells within a target organ, like, for example, tumor cells and/or vascular cells of tumor blood vessels. Moreover, this invention provides a method where, upon internalization of the previous-mentioned system by the target cells, triggered release at a high rate of the associated agent takes place, permitting efficient intracellular delivery and, thus, increased concentration of the transported cargo at the target site. Overall, this invention provides a method for the diagnosis, prevention and treatment of human diseases and disorders.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §120 of U.S.application Ser. No. 12/153,649, entitled “TARGETED DELIVERY TO HUMANDISEASES AND DISORDERS” filed on May 22, 2008, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the fields of human diseases anddisorders, more specifically to methods of selectively homing anddelivering an agent to human cells, combining targeting specificity withintracellular triggered release of the payload. The present inventioncan be applied, for example, in the treatment and/or diagnosis ofdifferent types of cancer as well as other diseases and disorders.

BACKGROUND INFORMATION

Amongst human diseases and disorders, cancer is the leading cause ofdeath throughout the western world. Even though, in most cases,diagnosis is often followed by surgery and the prognosis is favourable,recurrent forms often appear after surgery, indicating that metastaseswere already present by the time the disease was detected. This is themajor obstacle for a complete remission of the tumor.

Traditional chemotherapy constitutes one of the most important treatmentmodalities against cancer. Chemotherapeutic agents, upon systemicadministration, are generally characterized by a high volume ofdistribution that leads to a poor selectivity towards tumor cells andaccumulation in healthy tissues. Such pattern of distribution can leadto increased toxicities against normal tissues that also show enhancedproliferative rates, such as the bone marrow, gastrointestinal tract andhair follicles. Myelosuppression, alopecia or mucositis are examples ofsome of the most unpleasant and undesired consequences of fightingcancer with conventional therapy (Ferrara, 2005). Side effects thatoccur as a result of toxicities to normal tissues mean that anticancerchemotherapeutics are often given at sub-optimal doses, resulting in theeventual failure of therapy. This is often accompanied by thedevelopment of drug resistance and metastatic disease. Targeted drugdelivery towards tumor cells, on the other hand, offers the possibilityof overcoming these consequences by directing and concentrating thetherapeutic agent only at the desired target site, increasingtherapeutic efficacy through increased tumor cell death and decreasedincidence of side effects in healthy tissues (Allen, 2002).

Tumor cells require a dedicated and effective blood supply, which cannotbe provided by the existing vessels in normal tissues (Folkman, 1990).Therefore, angiogenesis, a process common on wound healing, starts todevelop in order to create an appropriate blood vessel network toirrigate the novel cellular mass. Since angiogenesis is controlled bypro- and anti-angiogenic factors, it appears to be a promising target incancer therapy (Ferrara, 2005). Angiogenic vessels present distinctfeatures at different levels, mainly on the markers expressed at thecell surface (Carmeliet, 2003). Many of these tumor vessel markers areproteins associated with tumor-induced angiogenesis and some arespecific for certain tumors (Pasqualini, 2002). Targeting therapeuticagents to the vasculature of tumors, as opposed to the tumor cellsthemselves, offers some additional advantages: eliminating tumor's bloodsupply can profoundly suppress tumor growth; blood vessels are morereadily accessible to intravenously administered therapy than tumorcells, and although tumor blood vessels acquire a tumor-associated‘signature’, they are composed of normal cells that do not readilyacquire mutations that could further lead to drug resistance (Boehm,1997); in addition, tumor vascular targeting avoids problems associatedwith intrinsic drug resistance such as those related with poor drugpenetration into a tumor due to high interstitial pressure gradientswithin tumors (Feron, 2004). Treatment selectivity against proliferativetumor-derived endothelial cells and minimal toxicity is likely to beachieved because angiogenesis in the adult is limited to wound healing,ovulation, pregnancy and atherosclerosis (Folkman, 2007; Folkman, 2005;Hanahan, 1996). In general terms, treatment selectivity can be achievedby designing a system where the agent is concealed, whereas the surfaceis decorated in a way that it has the ability to direct the system tothe target site, taking advantage of one or more distinct features ofthe pathological site. In this regard, one of the most importantstrategies in molecularly guided cancer pharmacology is the developmentof techniques that can modify the kinetic features of drugs byencapsulating them in nanosystems, like liposomes.

The development of physically and biological stable liposomes, composedwith a hydrophilic polymer, like poly(ethylene glycol), PEG, on itssurface, with an average size of 100 nm and containing chemotherapeuticdrugs, such as doxorubicin, was a significant achievement that,presumably, will have a great impact in the future of nanotechnology,within the field of human health. Coating the surface of liposomes witha hydrophilic polymer like PEG, strongly contributes to the formation ofa hydrophilic cloud around the liposomes (Needham, 1992 #44; Woodle,1992 #39; Hristova, 1995 #45; Hajitou, 2006 #62). Upon intravenousinjection, such hydrophilic shell dramatically decreases the rate andthe extent of electrostatic and hydrophobic interactions between thesurface of liposomes and blood components that mediate liposomal bloodclearance and/or disintegration (Lasic, 1991; Needham, 1992; Torchilin,1994; Woodle, 1992). The ability of drug-loaded PEG-grafted liposomes tolong circulate in blood, favours their accumulation in solid tumors (Wu,1993). Such accumulation, as well as that of macromolecules or polymericdrugs is greatly enhanced in tumor tissue relative to that in healthytissues, a phenomenon known as Enhanced Permeability and Retention,being generally observed in viable and rapidly growing solid tumors(Maeda, 2001). This phenomenon is supported by an extensive angiogenesisand impaired lymphatic drainage at the tumor interstitium (Maeda, 2000).Tumor vessels possess irregular cellular lining composed ofdisorganized, loosely connected, branched or overlapping endothelialcells, which contribute to tumor vessel leakiness (Carmeliet, 2003;Hashizume, 2000). As an example, it was previously demonstrated thatPEG-grafted liposomes, following transendothelial transport through gapsbetween endothelial cells, presented significant extravascularaccumulation in tumors (Yuan, 1994). Further improvements in theselective toxicity of anti-proliferative drugs might be achieved bycoupling ligands selective for the target cell to the liposome surface.Relatively few ligand molecules per lipo some (10-20) are required toselectively deliver high payloads of drugs to target cells via themechanism of receptor-mediated internalization (Allen, 2002). Unlikeother delivery systems such as drug

immunoconjugates or -immunotoxins, which deliver few molecules of drugor toxin (<10) per antibody (or immunotoxin) molecule, ligand-targetedliposomes can be exploited to deliver thousands of molecules of drugusing few tens of molecules of ligands covalently coupled on theliposome surface (Sapra, 2003). Coupling a ligand to a support should bea simple, fast, efficient and reproducible method, yielding stable,non-toxic bonds. Moreover, the coupling reaction should not alter thedrug loading efficiency, drug release rates, nor the biologicalproperties of the ligands, e.g. target recognition and bindingefficiency (Papahadjopoulos, 1991).

The versatility of liposomes as a delivery system allows the control ofthe location (spatial delivery) as well as of the rate of release(temporal delivery) of the transported agent. pH-sensitive liposomesconstitute a typical example where both spatial and temporal deliverycan be achieved. They are usually composed of a neutral cone-shapedlipid like dioleoylphosphatidylethanolamine (DOPE) and a weakly acidicamphiphile, such as cholesteryl hemisuccinate (CHEMS), and designed toform a stable lipid bilayer at neutral or basic pH but to rapiddestabilize in an acidifying endosome (Fonseca, 2005). SincepH-sensitive liposomes can facilitate cytosolic release of membraneimpermeable molecules, it might be feasible to combine their use with atargeting ligand that promotes receptor-mediated endocytosis. Overall,the development of a sterically stabilized pH-sensitive nanosystemcovalently coupled to a targeting ligand, able to target specific cells,like tumor cells and/or endothelial cells existing in tumor bloodvessels, containing a chemotherapeutic drug, such as doxorubicin, canhave a major impact on the therapeutic index of the encapsulatedpayload, in the treatment of diseases like human breast cancer.

SUMMARY OF INVENTION

The present invention provides a nanosystem with a pH-sensitive lipidcomposition (or incorporating a pH-sensitive disrupting agent),encapsulating an agent (like a drug or a diagnostic compound), and armedon the surface with a targeting ligand. Such a system, upon systemicadministration, for example, has the ability to target specific cellpopulations like tumor cells and/or endothelial cells existing in tumorblood ‘vessels. Upon reaching the target cells, the nanosystem iscapable of binding the target cells and is internalized throughreceptor-mediated endocytosis, where the acidification of the milieutriggers the destabilization of the nanosystem. Such destabilizationleads to release at a high rate of the associated payload to permitefficient intracellular release and, thus, increased concentration ofthe transported cargo at the target site. This versatile technologyallows the replacement of the lipid composition, of the encapsulatedpayload and of the ligand, depending on the purpose (treatment ordiagnostic) and/or the type of disease.

Overall, this invention provides, as a major benefit, improvedtherapeutic or diagnostic activity through specificity of action andtriggered release of the encapsulated payload (at the level of thetarget cell(s)), as well as reduced adverse side-effects. The field ofapplication includes therapy or diagnostic for cancer and for otherdiseases including, but not limited to inflammation, infectious diseasesor eye-diseases and disorders.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1—Cellular association of several formulations of liposomes withhuman breast cancer cells or human microvascular endothelial cells.Rhodamine-labelled non-targeted (SL), F3-targeted (SLF3), or liposomestargeted with a non-specific peptide (SLNS), at 1.2 mM total lipid/well,were incubated with 106 of (A) human tumor breast (MDA-MB-435S) or (B)human microvascular endothelial (HMEC

1) cell lines, at 4° C. or 37° C. for 1 h. Data are expressed as nmol oftotal lipid/mg protein, and are the mean of four experiments, each donein triplicate.

FIG. 2—Cellular association of several formulations of liposomes withspecific and non-specific cell lines. Rhodamine-labelled SL or SLF3 wereincubated with 106 of specific (MDA-MB-435S or HMEC-1) or non-specific(MCF-7 or TSA) cell lines, for 1 h at 4° C. or 37° C. The results areshown as the mean value of triplicates from a representative experiment.

FIG. 3—Competitive inhibition assay of cellular association of SLF3 withsPecific cell lines. One million of (A) human tumor breast (MDA-MB-435S)or (B) human microvascular endothelial (HMEC-1) cells were pre-incubatedwith 50 01 of free F3 peptide or without the peptide for 30 min at 37°C., followed by 1 h incubation with rhodamine-labelled SLF3 (at 0.8 mMTUwell) at 4° C. or 37° C. The results are shown as the mean value oftriplicates from a representative experiment.

FIG. 4—Cellular association of several formulations of liposomes withhuman breast cancer cells or human microvascular endothelial cellsanalysed by flow cytometry. One million of (A) human tumor breast(MDA-MB-435S) or (B) human microvascular endothelial (HMEC-1) cells wereincubated with SL (green), SLF3 (pink), or SLNS (blue) liposomeslabelled with rhodamine or encapsulating calcein at a concentration of0.6 mM TUwell, for 1 h at 4° C. (dotted line) or 37° C. (solid line).Cells were detached with dissociation buffer, washed with phosphatebuffer saline pH=7.4 (PBS) and immediately run in a FACSscan (BectonDickinson) for detecting cell aSsociated rhodamine or calcein. Thedifference between cellular association of SLF3 rhodamine-labelledliposomes at 37° C. and 4° C. is represented by a dotted purple line.

FIG. 5—Effect of several endocytosis inhibitors on the cellularassociation of SLF3 with (A) MDA-MB-4535 or (B) HMEC-1 cells. Onemillion of (A) human tumor breast (MDA-MB-435S) or (B) humanmicrovascular endothelial (HMEC

1) cells were pre-incubated with 0.45 M sucrose medium, 200 pM genisteinand 0.03 p.M wortmannin and 50 pM LY-29400 for 30 min at 37° C., for thepurpose of inhibiting clathrin- and caveolae-mediated endocytic pathwaysand macropinocytosis, respectively. Afterwards, cells were incubatedwith rhodamine-labelled SLF3 (0.2 mM TL/well for 1 h) or with thecorresponding control of each pathway: Alexa-Fluor

Transferrin (0.05 mg/ml for 30 min), BODIPYS-LactoCer (0.5 mM for 10min) or FITC

Dextran (10 mg/ml for 1 h) to evaluate the efficacy of the inhibition ofclathrin- and caveolae-mediated endocytic pathways and macropinocytosis,respectively. Cellular association of each control andrhodamine-labelled SLF3 was also performed at 4° C. withoutpre-treatment with inhibitors. Data are shown as means+/−S.D., based ontriplicates of at least two independent experiments.

FIG. 6—Cellular association of several formulations of liposomes withhuman breast cancer cells or human microvascular endothelial cellsanalysed by confocal microscopy. (A) Human tumor breast (MDA-MB-4355) or(B) human microvascular endothelial (HMEC-1) cells were incubated withnon-pH-sensitive (1) SLF3, (2) SL, (3) SLNS or with pH-sensitive (4)SLF3, (6) SL, (6) SLNS liposomes labelled with rhodamine andencapsulating calcein at a concentration of 0.6 mM TL/well, for 1 h at37° C. (upper row) or 4° C. (lower row). Cells were washed with PBS,fixed in 4% paraformaldehyde, mounted on Movioll' mounting medium(Calbiochem) and visualized with a LSM-510 laser-scanning confocalmicroscope (Carl Zeiss LSM510 Meta, Zeiss). All instrumental parameterspertaining to fuorescence detection and images analyses were heldconstant to allow sample comparison.

DETAILED DESCRIPTION OF INVENTION

The present invention provides a nanosystem combining a targetingability to specific cell subsets (like tumor cells and/or vascularcells), achieved by coupling an internalizing targeting ligand at thenanosystem surface, and triggered intracellular release (upon activationby acidification of the milieu) of the encapsulate payload at a highrate.

As shown herein, the term “payload” or “agent” means the portion of agreater whole which is distinct from the packaging required to transportit.

The term “ligand” designates the molecule linked to a support that iscapable of specifically directing a system to a target. The targetligand of the invention can be an aptamer, an antibody or a fragmentthereof (anti-CD19 that targets CD19 on lymphoma and multiple myelomacells (Sapra, 2003), anti-CD31 or PECAM-1 which recognises the humanCD31 cell surface antigen, anti-HER2-Fab which binds to HER2,bevacizumab that targets the vascular endothelial growth factor (VEGF),cetuximab used against epidermal growth factor receptor (EGFR)), aprotein (transferrin) a peptide (antagonist G that targets vasopressin,RGD4C for av integrins, CPRECES for aminopeptidase A, CNGRC for CD13,CKGGRAKDC that homes to white fat vasculature by targeting prohibitin,KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO:1) (F3), that binds tonucleolin (Hajitou, 2006)). The term “peptide” is used broadly todesignate peptides, fragments of proteins and the like, while“peptidomimetic” is used to mean a peptide-like molecule that has thebinding activity of the homing peptide, including compounds that containchemical modifications, non

naturally occurring aminoacids, peptoids and the like.

The ligand is susceptible of being identified based on its ability tohome to a specific organ, like a tumor, but not to the correspondingnon-target tissue.

The terms “home”, “selectively home” or “specific binding” mean that thetargeted system interacts with the target organ, on a ligand- and on acell-specific manner, after being administered to the subject.

As used herein, the term “tumor” includes tumor parenchymal cells,supporting stroma and angiogenic blood vessels that infiltrate the tumorcell mass. The terms “normal” or “non-tumor” are used to refer a tissuethat is not a “tumor”.

“System” or “conjugate” refers to the combination of interdependententities, herein presented as the agent, support and ligand, to form anintegrated whole with the ability to interact with a target organ orcell.

The physical, chemical or biological material linked to a homingmolecule is designated as “support” and encloses an agent to be targetedto a specific cell. Examples include liposomes, virus containing anagent such as a drug or a nucleic acid, non-toxic and biodegradablemicrodevices, microcapsules or a microbed composed of plastic, agarose,gelatine or other biological or inert material; a micelle, lipidmicelle, nanosphere, microsphere, lipid disc. Preferably the deliveryvehicle that acts as a support is a liposome composed of but not limitedto fully hydrogenated soy phosphatidylcholine, methoxy-polyethyleneglycol phosphatidylethanolamine, maleimide-polyethylene glycolphosphatidylethanolamine, N-methylpalmitoyloleoylphosphatidylcholine,phosphatidylserine, phosphatidylcholine,palmitoyloleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidylcholine, diphytanoylphosphatidylcholine,sphinomyelin, phosphatidylglycerol, dioleoylphosphatidylethanolamine,cholesteryl hem isuccinate, cholesterol, or a combination thereof. Thesupport should be non-toxic to the normal expression of the cell surfacemolecules and to the normal physiology of the subject and must notocclude circulation. If the subject used for study is not to besacrificed to collect a tumor or normal tissue, support should bebiodegradable as well.

The term “molecule” is used herein to designate a polymeric or anon-polymeric organic chemical, a nucleic acid or an oligonucleotide, apeptide or a peptidomimetic or a protein, antibody, growth fragment or afragment thereof presenting a linear or cyclic conformation, or anon-naturally occurring compound.

The invention provides a system composed of a support enclosing anagent, linked to a ligand that enables the selective and specifictargeting towards overexpressed molecules on the target cell, in orderto diagnose, treat or correct a disease and/or a disorder.

The ligand of the invention is linked to the surface of the support in away that the specific sequence is able to interact with the targetmolecule overexpressed on the surface of a specific cell. An appropriatespacer, with or without a reactive group, can be positioned between theligand and the support, in such a way that the mentioned interaction isnot hindered. Conjugation of PEG to the peptide requires a suitablefunctional group at the end of the PEG and the N-terminal of thepeptide. When an amine (-peptide-NH-PEG) is the linkage functionalgroup, a PEG with an end-group functionalized by a halide (e.g. —CI,—Br, and —I) or a sulfonate (e.g. -0S02 Ca H4 CH3, -0S02 CH2 CF3) can beused to couple with the amino group at the N-terminal. When a urethane(peptide-NHC(0)0-PEG) is the linkage functional group, a PEG with anend-group functionalized by an active carbonate (eg. —C(0)-Im,-0C(0)-pNP, —OC(0)-NHS, -0C(0)-TCP) can be used to couple with the aminogroup at the N-terminal. When an amide (peptide-NHC(0)-PEG) is thelinkage functional group; a PEG with the end-group functionalized by theactivated carboxyl group (e.g., the carboxyl group activated byDCC/HOBt, DCC/dimethylaminopyridine (DMAP), DIPCDI/HOBt, and EDC/NHS)can be used to couple with the amino group at the N

terminal. When a thio ester (peptide-C(0)CH2 SC(0)-PEG) is the linkagefunctional group, a PEG with the end-group functionalized by the thioacid (-PEG-C(0)S) can be used to couple with the N-terminal modified tobromoacetyl (peptide-C(0)CH2 Br). When a thio ether (peptide-C(0)CH2SCH2-PEG) is the linkage functional group, a PEG with the end-groupfunctionalized by the thiol group (-PEG-CH2 SH) can be used to couplewith the N-terminal modified to bromoacetyl (peptide-C(0)CH2 Br). Whenthe thioether of a maleimide/thio conjugate is the linkage functionalgroup, a PEG with the end-group functionalized by a thiol group(C(0)-PEG-C(0)CH2 CH2 SH) can be used to couple with the N-terminalmodified to the maleimide group (maleimide-CH2 CH2 C(0)-peptide)(Zalipsky, 1995).

Primary amines, present at the N-terminal of a biomolecule, can bemodified with the introduction of SH groups. In a preferred embodimentof the present invention, this modification can be achieved usingTraut's reagent (2-Iminothiolane hydrochloride). Thiol groups are ableto react directly with maleimide present on the sUpport surface leadingto a stable thioether bond. Under these circumstances, immobilization ofa thiolated peptide creates a spacer of five atoms length (maleimide

S—CH2CH2C(0)NH-peptide).

The reactive group present in the spacer not only can be an efficientmeans of linking the ligand to the support, but also can contain a tagto facilitate recovery or identification of the system. Identificationof the ligand or the support linked to the ligand with a known label,allows in vitro cells or in vivo organs or tissues to be collected,molecules recovered and compared with the control cell population, organor tissue. The term “control cell, organ or tissue” means a cell, organor tissue other than the one for which the identification of the homingmolecule is desired.

The conjugate can be multivalent, presenting more than one homing ligandthat selectively homes to the designated molecule(s) on the targetcells. The conjugate can be directed to the target cell by an externalligand covalently linked to its surface directly or through a reactivegroup inserted in the support. The homing molecule of the invention canbe linked to other supports besides liposomes, such as a physical,chemical or biological delivery systems or a cell, upon administration.Also, according to the method of the invention, a variety of therapeuticagents can be directed to tumor blood vessels and tumor cells in asubject.

Doxorubicin (DXR) is a chemotherapeutic agent widely used in cancertherapy with anti-angiogenic properties (Devy, 2004). Otherchemotherapeutic agents successfully used include alkylating drugs, suchas cyclophosphamide, chlorambucil, melphalan, busulfan, lomustine,carmustine, chlormethine (mustine), estramustine, treosulfan, thiotepa,mitobronitol; cytotoxic antibiotics, such as doxorubicin, epirubicin,aclarubicin, idarubicin, daunorubicin, mitoxantrone (mitozantrone),bleomycin, dactinomycin and mitomycin; antimetabolites, such asmethotrexate, capecitabine, cytarabine, fludarabine, cladribine,gemcitabine, fluorouracil, raltitrexed (tomudex), mercaptopurine,tegafur and tioguaninc; vinca alkaloids, such as vinblastine,vincristine, vindesine, vinorelbine and etoposide; other neoplasticdrugs, such as amsacrine, altetarmine, crisantaspase, dacarbazine andtemozolomide, hydroxycarbamide (hydroxyurea), pentostatin, platinumcompounds including: carboplatin, cisplatin and oxaliplatin, porfimersodium, procarbazine, razoxane; taxanes including: docetaxel andpaclitaxel; topoisomerase I inhibitors including inotecan and topotecan,trastuzumab, and tretinoin; SN-38, ET-743, TLK 286; anti-inflammatoryagents: ibuprofen, aceclofenac, acemetacin, azapropazone, celecoxib,dexketoprofen, diclofenac sodium, diflunisal, cetodolac, fenbufen,fenoprofen, flubiprofen, indomethacin, acetaminocin, piroxicam,rofecoxib, sulindac, tenoxicam, tiaprofenuic acid, aspirin andbenorilate; antiangiogenic agents or angiolytic agents such as but mitlimited to: Angiostatin (plasminogen fragment), antiangiogenicantithrombin III, Angiozyme, ABT-627, Bay 12-9566, Benefin, Bevacizumab,BMS-275291, cartilage-derived inhibitor(CDI), CAI, CD59 complementfragment, CEP-7055, Col 3, Combretastatin A-4, Endostatin (collagenXVIIIfragment), Fibronectin fragment, Gro

beta, Halofuginone, Heparinases, Heparin hexasaccharide fragment,HMV833, Human chorionicgonadotropin (hCG), IM-862,Interferonalpha/beta/gamma, Interferon inducible protein (IP-10),Interleukin-12, Kringle 5 (plasminogen fragment), Marimastat,Metalloproteinase inhibitors (TIMPs), 2-Methoxyestradiol, MMI 270 (CGS27023A), MoAbIMC-1C11, Neovastat, NM-3, Panzem, PI-88, Placentalribonuclease inhibitor, Plasminogen activator inhibitor, Plateletfactor-4 (PF4), Prinomastat, Prolactin16 kD fragment, Proliferin-relatedprotein (PRP), PTK 787/ZK 222594, Retinoids, Solimastat, Squalamine, SS3304, SU 5416, SU6668, SU11248, Tetrahydrocortisol-S,Tetrathiomolybdate, Thalidomide, Thrombospondin-1(TSP-1), TNP-470,Transforming growth factor-beta (TGF-b), Vasculostatin, Vasostatin(calreticulin fragment), ZD6126, ZD 6474, Farnesyl transferaseinhibitors (FTI), Biphosphonates. Porphyrins are also widely used incancer treatment, as well as in the treatment of ocular diseases anddisorders, within photodynamic therapy. Thus, the targeted drug can be acytotoxic, an anti-cancer, an anti-inflammatory, an anti-angiogenic, anangiolytic, a vascular disrupting agent or an agent for photodynamictherapy. The drug or a combination of two or more drugs can beencapsulated, entrapped, intercalated within the core or associated withthe delivery vehicle.

The conjugate should have a diameter comprised between 100 and 200 nm tobetter achieve the final purpose of encountering the target cell throughthe circulation by passing relatively unhindered through the capillarybeds without occluding circulation (Drummond, 1999). The system isadministered to the subject, which can be a vertebrate, such as amammal, particularly a human, and passes through the tumor and itsvasculature where it specifically interacts with the target cells.

Targeting selectivity depends on the overexpression of a cell surfacereceptor on only one or a few cell types. Controls can be a similarpeptide lacking the binding sequence or a different cell line notoverexpressing the receptor to the target ligand presented in exampleII.

A conjugate to selectively bind the target cell must overcome obstaclessuch as long diffusion distances, tight cell adherence, a dense fibrousstroma and the high interstitial pressure gradient towards the interiorof the tumor mass (McDonald, 2002). These are the reasons why vasculartargeting also emerges as an agreeable option, since endothelial cellsare easily accessible to a circulating conjugate. Moreover, angiogenicvessels present distinct features at different levels, such asstructurally irregular walls, typically poor and abnormal blood flow andleakiness (Carmeliet, 2003; Pasqualini, 2002). Overall, these featurescan facilitate, on one hand, tumor metastization and, on the other hand,the therapeutic agent to reach the tumor. By affecting the ability of atumor vascular network to organize, additional gains are expected, forexample, and in the case of anticancer therapy, on the primary tumor andalso on metastases. Moreover, cellular receptors express a conservativenature, which means that tumor and vascular cells, namely endothelialcells, can share the same surface markers. Thus, a tumor homing moleculethat binds a target molecule in the tumor vasculature of a mouse is alsocapable of binding to the corresponding target molecule in the tumorvasculature of a human or other mammalian. In fact, the same targetmolecule can be shared by different tumors (Folkman, 1997). However, forthe final purpose of administering a therapeutic conjugate to a subject,such as a mammal, one must possess pharmaceutical acceptable propertiesaccording to the route of administration and target location. Severalsystems, like liposomes, are non-toxic, biocompatible, easily made andadministered, which are advantageous characteristics for parenteraladministration in an effective amount to permit therapeutic, diagnosticor detecting effect. The term “effective amount” refers to the necessaryquantity for producing the desired action.

The conjugate can incorporate a chemotherapeutic or cytotoxic agent, adiagnostic agent, a detecting agent or a gene therapeutic agent. Thecontent of the conjugate can be incorporated or encapsulated in apassive or active manner and the delivery mechanism can be controlled byengineering the system composition. The tumor microenvironment exhibitsunique features that can be advantageous to promote disruption of thesystem and its content release. Poor vascular organization and impairedlymphatic drainage lead to the accumulation of products of cellularmetabolism, such as lactic and carbonic acid, which lowers theextracellular milieu pH value (Gatenby, 2004). The same happens in theendosomal pathway, where disruption and content release of the conjugatetake place in early and late endosomes (Simoes, 2004).

Liposomes can be composed of weakly acidic amphiphiles and neutralcone-shaped lipids to allow control over the disruption of the lipidmembrane. At physiological pH, stable liposomes are formed, butacidification triggers protonation of the carboxylic groups of theamphiphiles, such as CHEMS (Straubinger, 1993), reducing theirstabilizing effect and thus leading to liposomal destabilization, sinceunder these conditions, cone-shaped molecules likephosphatidylethanolamine (PE) revert into their inverted hexagonalphase. The choice of the amphiphilic stabilizers as well as its molarpercentage with respect to the PE content are imposed by the desiredproperties of the liposomes, including the extent of cellularinternalization, the fusogenic ability, pH-sensitivity and stability inbiological fluids (Drummond, 2000).

A more rapid rate of drug release from endosomes or lysosomes, leads tomore rapid delivery of the drug to its intracellular site of action,resulting in improved therapeutic efficacy (Ishida, 2006).

In the example illustrated herein, the receptor that specifically bindsthe tested ligand has been identified as nucleolin (Christian, 2003).

Nucleolin is a ubiquitous, nonhistone nucleolar phosphoprotein ofexponentially growing eukaryotic cells. It is described as having a rolein controlling the organization of nucleolar chromatin, packaging ofpre-RNA, rDNA transcription and ribosome assembly. Therefore, it isimplicated in proliferation and growth, cytokinesis, replication,embryogenesis and nucleogenesis. The protein appears in cells beforetranscription and ribosome synthesis starts (Srivastava, 1999).

The nucleolin polypeptide consists of a negatively charged NH2-terminaldomain, an RNA-binding domain and a COOH-terminal domain rich in RGGmotifs. The first region has an analogous high-mobility group function.Phosphorilation of this domain enhances nucleolin degradation byproteases, compromising its stability, but it is crucial for rDNAtranscription. The central globular domain is involved in pre-RNArecognition, condensing and packaging in the nucleolus. Thecarboxyl-terminal domain controls nucleolin interaction with ribosomalproteins by permitting RNAs access to the RNA binding motifs located inthe central region of nucleolin. Both amino and carboxyl terminals canbe regulated by proteolysis. Cleaved nucleolin activates autolyticendonucleases, which fragment DNA to cause apoptosis. In proliferatingcells, the expression of a proteolytic inhibitor prevents nucleolin'sself-degradation. Therefore, the levels of this protein are elevated intumor and other rapidly dividing cells (Srivastava, 1999).

A homing molecule for nucleolin can be an anti-nucleolin antibody or ahigh mobility group protein (HMGN-2) derived peptide, such as the31-amino acid fragment that corresponds to the sequenceKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO:1) (F3), identified by phagedisplay libraries (Porkka, 2002). F3 binds to the NH2-terminal domain ofnucleolin and can accumulate preferentially in angiogenic vasculature oftumors as compared to non-tumor vessels. Since nucleolin is present inthe cell surface in a phosphorilated form and in the nucleus membrane,the peptide can be internalized by its specific target cells and betransported to the nucleus. It is recognized that the F3 peptide shownherein is useful for tumor and endothelial cell homing (Porkka, 2002)and that it can be attached to a support to form a conjugate that cancarry a payload such as a chemotherapeutic agent, a diagnostic agent oran imaging agent directed to cells involved in tumor growth.

As shown in Example 1, for active encapsulation of the lipid-basedsystem covalently coupled with the F3 peptide, the mixture ofliposome-forming lipids is dried to form a thin film and hydrated withan aqueous medium containing the solute species that will form theaqueous phase in the interior of the vesicle. Afterwards, those speciesare removed and the drug is added to the exterior of the liposome forremote loading. Methods such as size exclusion chromatography, dialysisor centrifugation can be applied to remove the non-encapsulated drug. Inanother embodiment, a trapping agent can be included in the interior ofthe liposome to complex with the encapsulating agent and lead to itsretention.

The use of lipids with high transition temperatures(distearoylphosphatidylcholine (DSPC); hydrogenated soyphosphatidylcholine (HSPC)) and the incorporation of cholesterol (CHOL)and lipid conjugates such as distearoylphosphatidylethanolaminepolyethylene glycol (DSPE-PEG), lead to a significant decrease ofleakage of the encapsulated drugs during blood circulation or in theextracellular milieu. Moreover, such lipids also reduce non-specificinteractions between the liposomes and serum proteins (opsonins), thuspreventing liposome clearance by the cells of the reticuloendothelialsystem (RES), increasing circulation time for optimizing the interactionof the system with the target cells (Allen, 1987; Gabizon, 1992).

Size of liposomes can be engineered by forcing the passage of thevesicles through appropriate pore size membranes in an extruder. It isadvised for liposomes to have a size small enough to allow its passagethrough capillaries and to extravasate from the vascular compartment iftumor cells are to be targeted (Yuan, 1994).

F3, used as an example in this description, is coupled to the surface ofthe liposome by an imidoesther link between the thiol group of thederivatized peptide and the maleimide group on the surface of thesupport. Conjugating a peptide to a support can potentially affect thesystem's homing capacity. As shown herein, peptide specificity andability to home to the target receptor are not affected (Examples II andIII). It is important to emphasize that the encapsulation of an agent isalso susceptible to compromise its desired action. In the present case,it is demonstrated that the encapsulated agent maintains itscharacteristic features and action (Examples II and III).

Cellular internalization studies were performed by comparison of cellsincubated with target or non-targeted conjugates at internalizationpermissive (37° C.) and non-permissive (4° C.) temperatures. Resultsconfirm the importance of the ligand in the recognition of the system bythe target cells. Moreover, other cell lines, such as MCF-7 and TSA showno difference between the internalization levels of the targeted and non

targeted conjugate, which indicates that the system is capable of homingto a specific tumor and tumor vascular endothelial cells. Specificity ofthe targeting ligand is confirmed by a competitive binding assay(Example II).

The mechanisms by which a system enters into a cell are distinct andusually divided into two broad categories: phagocytosis, a processrestricted to specialized mammalian cells, and pinocytosis, which occursin all mammalian cells and comprehends macropinocytosis,clathrin-mediated endocytosis, caveolae-mediate endocytosis, as well asother less characterized clathrin- and caveolae-independent endocyticpathways (Conner, 2003).

As shown in Example III, internalization into the cells is stronglyenergy-dependent and the clathrin-mediated endocytotic pathway appearsto be the most probable portal of entry of the conjugate into tumor andendothelial cells of angiogenic blood vessels.

Confocal microscopy observations corroborate the results frominternalization studies (Example II). Non-targeted controls show nostaining for the lipid marker as opposed to the F3-targeted samples.Cells incubated with ligand targeted pH-sensitive liposomes show a moreevident green intracellular staining than targeted non-pH-sensitiveliposomes. Such results indicate that therapeutic gains can be expectedwith a targeted formulation capable of promoting intracellular triggeredrelease of an encapsulated agent to both tumor and endothelial cells ina subject.

The antitumor and the anti-angiogenic activity of doxorubicin make it agood model to demonstrate the potential of the invented technologicalplatform. As an example, doxorubicin is encapsulated in a lipid-basednanosystem, covalently coupled to a peptide, and incubated with breastcancer and endothelial cells in different ranges of drug concentrationduring predetermined times (Example IV).

Results demonstrate that the cytotoxic activity of the drug delivered bythe targeted system of the invention is increased againstnucleolin-overexpressing cells as compared to nucleolinnon-overexpressing cells. Incubation of cells with a system composed ofa therapeutic agent and a target homing molecule is more effective incausing cell death than incubation with the system without the specificligand. Cytotoxicity of doxorubicin-containing peptide-targeted lipidconjugates against MDA

MB-435S (breast ductal carcinoma cells) and HMEC-1 (human mammaryepithelial cells) was compared in vitro to free doxorubicin,doxorubicin-containing non-targeted conjugates or conjugates targeted bya non-specific peptide. Results demonstrate that selective accumulationof the conjugate increases toxicity of the agent, reducing the dosagerequired for inducing 50% of cell death.

The system's unique composition allows specific interaction with targetcells along with programmed intracellular delivery of the payload,resulting in higher efficiency of action of the transported agent. Thesefeatures are congregated for the first time on the same technologicalplatform, differentiating it from those reported in the literature.

Documents WO 2005/094383, WO 2000/023570, WO 2007/039783, WO2003/084508, WO 2001/085093 and WO 1998/016201 refer to lipid-basednanovesicles with the ability to promote triggered release, upon anadequate stimulus. Nevertheless, the system described herein is uniquein terms of its lipid composition and targeting specificity, mechanismof payload release and has the additional advantage of exhibitingadequate properties for intravenous administration such as: prolongedblood circulation times due to reduced non-specific interactions withserum proteins (opsonins), thus preventing liposome clearance by thecells of the reticuloendothelial system (RES); a diameter small enoughto avoid emboli or stroke, upon administration, and to facilitateextravasation from blood vessels into the tumor mass.

Document WO 2003/087124, although it mentions the F3 ligand to exemplifythe targeted nanosystem herein, it does not include lipid-basednanovesicles with the ability to promote intracellular triggered releaseof the associated payload.

Document WO 2007/100904 refers to F3 peptide as one of the putativespecific ligand of a L-methionase-drug conjugate which does not have theability to be internalized by tumor vasculature endothelial cells and isnot a lipid-based system.

Document WO 2005/019429 refers to two different levels of cellulartargeting: tumor and endothelial cells. However, the purpose of thatinvention (enhancing phagocytosis or phagocyte activity) is fairlydifferent from the one described herein (enhance receptor-mediatedendocytosis to diagnose, treat or correct a disease and/or a disorder).

The present invention satisfies the need for selectivity and programmeddelivery of a payload to specific cell populations within a targetorgan, like for example, tumor and/or vascular cells (endothelial cells,mural cells) of tumor angiogenic blood vessels, inhibiting, in the caseof a tumor, its growth, development and metastization.

EXAMPLES

The following examples are intended to illustrate but not limit thepresent invention.

Example I Preparation and Characterization of Different Lipid-BasedNanosystems

This example provides methods for encapsulating a payload such as achemotherapeutic agent into a lipid-based nanosystem and for coupling ahoming molecule such as peptide to its surface.

Non-pH-sensitive liposomes were composed of fully hydrogenated soyphosphatidylcholine, cholesterol, distearoylphosphatidylethanolaminemethoxy(polyethylene glycol) (2000); distearoylphosphatidylethanolaminemaleimide(polyethylene glycol) (2:1:0.06:0.04, molar ratio). The lipidfilm was hydrated at 65° C. in 250 mM ammonium sulphate solution pH 5.5.

The pH-sensitive formulation was composed ofdioleoylphosphatidylethanolamine, cholesteryl hemisuccinate, fullyhydrogenated soy phosphatidylcholine, cholesterol anddistearoylphosphatidylethanolamine methoxy(polyethylene glycol) (2000);distearoylphosphatidylethanolamine maleimide(polyethylene glycol)(4:2:2:2:018:0.12, molar ratio). The lipid film was hydrated at 65° C.in 250 mM ammonium sulphate solution pH 8.5.

Both formulations were extruded sequentially through polycarbonatemembranes of 0.2 and 0.1 pm pore size at 65° C. using a LipoFast miniextruder (Lipofast, Avestin) to obtain a uniform size distribution(Daleke, 1990). The buffer was exchanged in a Sephadex 0-50 columnequilibrated with 100 mM NaCH3COOH/70 mM NaCl pH 5.5 for thenon-pH-sensitive formulation and with 25 mM Trizmabase/10% sucrose pH 9for the pH-sensitive formulation. Doxorubicin was then incubated withliposomes for 1 h at 65° C., in the absence of light and encapsulated bythe ammonium sulphate gradient method (Bolotin EM, 1994). Freedoxorubicin was removed by running the liposomes through a Sephadex G-50column equilibrated with 25 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/140 mM NaClbuffer pH 7.4 (HBS) (for non-targeted liposomes), 25 mM HEPES/25 mM MES(2-(N

morpholino)ethanesulfonic acid)/140 mM NaCl pH 6.5 (for targetednon-pH-sensitive liposomes) or 25 mM HEPES/25 mM MES/140 mM NaCl pH 7.2(for targeted pH

sensitive liposomes). To further prepare targeted liposomes, thethiolated derivative of F3 peptide (Genosphere Biotechnologies) wasobtained by reaction of the peptide with 2-mercaptopropionimidatehydrochloride (a.k.a. 2-iminothiolane), at a 1:5 molar ratio in 25 mMHEPES/140 mM NaCl buffer (pH=8), for 1 h at room temperature. Liposomeswere then incubated overnight at room temperature with the activatedpeptide at a maleimide/activated peptide molar ratio of 1:2. Activationand coupling of F3 peptide took place in an inert N2 atmosphere insiliconcoated glassware (Sigmacote, Sigma) Free maleimide groups werequenched by incubation with an excess of 2-mercaptoethanol for 30 min atroom temperature. Uncoupled peptide was separated in a Sepharose CL-4Bcolumn equilibrated with HBS pH 7.4.

Final lipid concentrations were determined based on lipid phosphorousassay by Fiske and Subarrow (Bartlett, 1959). The encapsulateddoxorubicin was quantified by measuring UV absorbance at 492 nm. A finalconcentration of 120-180 119 of doxorubicin per ilmol of phospholipid(95% loading efficiency) was achieved. Loading efficiency was determinedusing the formula [(DXR final concentration/Total lipid finalconcentration)/(DXR initial concentration/Total lipid initialconcentration)]×100. The size of the liposomes varied between 100-150nm, measured by dynamic laser scattering with a Coulter submicronparticle size analyzer.

Example II Cellular Association Studies Cellular Culture

MDA-MB-435S and HMEC-1 cells were cultured in RPMI 1640 (Sigma)supplemented with 10% (v/v) heat-inactivated Foetal Bovine Serum (FBS)(Invitrogen), 100 U/ml penicillin, 100 1.19/ml streptomycin (Sigma)(full medium) and maintained within their exponential growth phase at37° C. in a humidified incubator (90% humidity) containing 5% CO2.HMEC-1 cells medium was also supplemented with 10 ng/ml mouse epidermalgrowth factor (EGF) and 1 p.g/ml hydrocortisone (Sigma). MCF-7 (ATCC)and TSA (ATCC) were grown in Dulbecco's Modified Eagle's Medium (DMEM)(Sigma), supplemented as described above (full medium).

Cellular Association Studies

Cellular association studies were performed by fluorimetry, flowcytometry and confocal microscopy.

Rhodamine-labelled liposomes (targeted, targeted by a non-specific (NS)peptide—ARALPSQRSR (SEQ ID NO:2) (Porkka, 2002)—or non-targeted), wereincubated with one million of tumor (MDA-MB-4355) or endothelial cells(HMEC-1) at 4 or 37° C. and within a lipid concentration range of0.1-1.2 mM of total lipid/well.

To assess cellular association by fluorimetry, cells were lysated andrhodamine's fluorescence was measured in the supernatant in a SpectraMaxGemini EM plate reader fluorimeter (Molecular Devices). Cellular proteinwas determined by the BCATP4 Protein Assay Kit (Pierce). Results wereexpressed as nmol of total lipid/mg of protein.

To determine cellular association and payload delivery by flowcytometry, cells were incubated with rhodamine-labelled or calceinloaded liposomes at 37 or 4° C., for 1 h, detached with dissociationbuffer, washed with phosphate buffer saline pH=7.4 (PBS) and immediatelyrun in a FACSscan (Becton Dickinson) for detecting cell associatedrhodamine (FL2-H) and calcein (FL1-H). A total of 40,000 events werecollected and files were analysed with Cell Quest Pro software.

At 37° C., the extension and the rate of cellular association anddelivery of the payload observed for peptide-targeted liposomes washigher than the one observed for non-targeted liposomes or liposomestargeted by a non-specific peptide, pointing out that the interaction ofthe former with the target cells was peptide-specific (FIG. 1). Theimproved cellular association revealed to increase with the increase ofthe lipid concentration (data not shown). The substantial increase inthe levels of association for peptide-targeted liposomes as thetemperature was raised from non-permissive (4° C.) to permissivetemperatures (37° C.) for endocytosis, suggested that they were beinginternalized (FIG. 1). Targeted and non-targeted liposomes presentedsimilar extensions of cellular uptake when incubated with non-specificcell lines like the TSA and MCF-7 cells, revealing that the interactionof F3-targeted liposomes was cell-specific (FIG. 2).

Further evidence to support the endocytosis of the targeted liposomescomes from the intracellular fluorescence observed in confocalexperiments performed at 37° C. as opposed to what happened at 4° C.,where little or no staining is observed.

Liposomes were double-labelled with rhodamine (red label of the lipidmembrane) and calcein (green label of the aqueous core). MDA-MB-435S orHMEC-1 cells were seeded on glass cover slips in 12-well flat bottomplates at a density of 2×105 cells per well. After complete adhesion,cells were incubated at 4 or 37° C. with the fluorescently

labelled liposomes for 1 h and washed three times with PBS, following byfixation with 4% paraformaldehyde in PBS during 20 min at roomtemperature. After washing with PBS, cells were mounted on Moviol®mounting medium (Calbiochem) and visualized with a LSM-510laser-scanning confocal microscope (Carl Zeiss LSM510 Meta, Zeiss),using a 488 nm and 561 nm excitation laser and a 63×/1.40 oil objective.Cells were optically sectioned and images (512×512 pixel) were acquiredusing the LSM-510 software. All instrumental parameters pertaining tofluorescence detection and images analyses were held constant to allowsample comparison.

Results from confocal microscopy on the intracellular uptake ofF3-targeted liposomes demonstrate that after 1 h incubation,rhodamine-labelled (red) liposomes, whether pH-sensitive ornon-pH-sensitive, are localized inside the target cells. In contrast,after incubation with non-targeted liposomes, no red fluorescence isobserved inside or outside the cells. Cells incubated with thenon-specific peptide for the target molecule overexpressed on the cellsurface, exhibit only a mild red fluorescence. These findingscorroborate the different levels of cellular rhodamine contentquantified by fluorimetry and flow cytometry in both tumor andmicrovascular endothelial cells incubated with the differentformulations. Moreover, a diffuse intracellular green staining isobserved upon incubation with calcein loaded pH-sensitive targetedliposomes at 37° C., as opposed to the punctuated staining observed withthe targeted non-pH-sensitive formulation, confirming an improvedintracellular release of the liposomal payload when delivered by theformer formulation.

Competitive Inhibition

Cells were plated on a 48-well flat bottom plate at a density of onemillion cells per well. After adherence, cells were pre-incubated 30 minwith free F3 peptide at a non-toxic concentration of 50 IAM at 4 or 37°C. Controls were incubated with culture medium. Afterwards, F3-targetedPEG-grafted rhodamine-labelled liposomes were added and furtherincubated for 1 h at the mentioned temperatures. Cellular associationwas assessed by fluorimetry as previously described.

The homing of the F3-targeted PEG-grafted liposomes to breast cancercells is inhibited when synthetic F3 peptide is pre-incubated with thetarget cells (FIG. 3), suggesting, in agreement with the results fromfluorimetry, flow cytometry and confocal experiments, that the system isbeing internalized by receptor-mediated endocytosis.

Example III Mechanisms of Cellular Uptake

Macropinocytosis, a distinct form of endocytosis, depends on a sodiumgradient and can be blocked by amiloride andN-ethyl-N-isopropylamiloride (EIPA), inhibitors of a Na+/H+ exchanger.It also depends on F-actin microfilament rearrangement and onphosphoinositide 3-kinase (PI3), a key enzyme in the downstreamsignaling of macropinocytosis. The inhibition of F-actin elongation bycytochalasin B and of PI3 by wortmannin and LY-294002 blocksmacropinocytosis efficiently (Rejman, 2005). Dextran labelled with FITC(FITC-Dextran, Sigma), used as a control for this pathway, was incubatedwith cells to evaluate the efficiency of the inhibition by wortmanninand LY-294002.

MDA-MB-4355 and HMEC-1 cells were seeded on 48-well plates at a densityof one million cells per well and allowed to adhere for 24 h.Afterwards, they were pre-incubated with the inhibitors for 30 min andsubsequently co-incubated with rhodamine

labelled SLF3 for 1 h. The cellular content of rhodamine of theinhibitor-treated groups was then compared with an inhibitor-freecontrol. Results were calculated according to the formula:(concentration of the label in inhibitor-treated cells/concentration ofthe label in inhibitor-free cells)×100.

As depicted in FIGS. 4 and 5, cytochalasin B does not decrease cellularcontent of rhodamine, whereas wortmannin and LY 294002 inhibition causea minor decrease of approximately 20% in both cell lines. Thisdemonstrates that macropinocytosis is not a major pathway regarding theintracellular accumulation of F3-targeted liposomes.

Caveolae-mediated endocytosis is another portal of entry into the celland is known to be inhibited by filipin and genistein (Rejman, 2005).BODIPY-lactosylceramide (BODIPY-lactocer, Molecular Probes) reported tobe exclusively internalized via caveolae-mediated mechanism (Puri,2001), was used as a control for the inhibition of this pathway.Genistein-mediated blockade of tyrosine kinases necessary for this typeof endocytosis results in a slight decrease in the cellular content ofrhodamine (12.2% of control for MDA-MB-435S and 7.87% for HMEC-1 cells).

In further experiments, clathrin-mediated endocytosis was blocked withhypertonic medium. Alexa-Fluor-Transferrin (Molecular Probes) was usedas the positive control to assess the efficiency of theprevious-mentioned inhibitors. A strong decrease of cellular content ofrhodamine (65.7% in MDA-MB-435S cells and 31% in HMEC-1 cells) isobserved after treatment with 0.45 M sucrose. These results corroboratethe observation previously-mentioned in example II that internalizationinto the cells is strongly energy dependent, since a decrease in theincubation temperature from 37 to 4° C. was accompanied by a 2.4-foldlowering of the cellular content of rhodamine. Furthermore, thesestudies indicate that F3 peptide-targeted liposomes are internalized bya receptor-mediated mechanism, most likely through the clathrin

mediated endocytosis pathway, which is reinforced by the results fromcompetitive inhibition described in example II, where cells previouslyincubated with the free peptide exhibited a decrease in the content ofrhodamine as opposed to control cells not incubated with the peptide.

Example IV Cytotoxicity Studies

In vitro cytotoxicity of free doxorubicin (DXR) and DXR-containingliposomes was determined for MDA-MB-4355 and HMEC-1 cells using the MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)proliferation assay (Mosmann, 1983). Briefly, cells were plated in96-well plates at a density of 8,000 cells per well and incubated withfree DXR, non-targeted pH-sensitive (or non-pH-sensitve) liposomescontaining DXR or targeted pH-sensitive (or non-pH-sensitve) liposomescontaining DXR. Additional controls included free peptide and emptytargeted liposomes. Cells were incubated for 1, 3, 24 and 48 h at 37° C.in an atmosphere of 95% humidity and 5% CO2. At the end of theincubation time, cells were gently washed twice with cold PBS to removeDXR and maintained in fresh medium for a total of 96 h. Afterincubation, the medium was replaced by a solution of 0.5 mg/ml MTT andthe cells were further incubated for 4 h at 37° C. in an atmosphere of95% humidity and 5% CO2. Crystals were dissolved in acidic isopropanoland absorbance in each well was read at 570 nm in a microplate reader(Multiscan EX—Thermo Electron Corporation). IC50 of DXR mediated by thedifferent formulations was determined from the dose/response curves.

The cytotoxicity of DXR, either free or encapsulated in PEG-graftedliposomes, was compared as a function of time. IC50 decreases as theexposure of cells to drug increases from 1 h to 48 h (Table 1). After 24h of incubation, SLF3[DXR] is at least 17-fold more cytotoxic thanSL[DXR] against MDA-MB-435S cells and 4.4-fold more cytotoxic againstHMEC-1 cells, suggesting that binding and internalization of thetargeted liposomes are contributing to the increased cytotoxicity. Thisobservation is reinforced by the non-cytotoxic nature of the emptyliposomes and by the fact that all of the lipid-based formulationstested showed minimal leakage in HBS and culture medium (data notshown). As demonstrated by confocal microscopy, the pH-sensitiveformulation allows a large amount of drug to become rapidlybioavailable, leading to an increased intracellular concentration ofDXR, which justifies the higher levels of cytotoxicity.

Free DXR has the highest levels of cytotoxicity against all the celllines in vitro, but it does not distinguish between targetreceptor-expressing and target receptor-nonexpressing cell lines. It isimportant to point out that the in vitro cytotoxicity results obtainedwith free drug do not take into account the unfavourablepharmacokinetics and biodistribution that doxorubicin presents in vivo(Gabizon, 1994). Because free DXR is rapidly and widely redistributed totissues after systemic administration, it is expected that thepH-sensitive targeted formulation, with their ability to selectivelybind the target cells and to efficiently deliver (intracellularly) theencapsulated payload, will have an advantage over the free drug in vivo.

Overall, treatment of human endothelial and tumor cells withpeptide-targeted pH-sensitive liposomes containing doxorubicin(SLF3(DXR] pH-sensitive), induces a faster and stronger inhibition ofcell growth than the other tested formulations containing doxorubicin(non-targeted pH-sensitive, SUDXR] pH-sensitive, or peptide-targeted ornon-targeted, non-pH sensitive liposomes, SLF31DXR] and SLIDXRJ,respectively) (Table 1). Targeted intracellular triggered-doxorubicinrelease (upon decrease of pH) proved to be a crucial feature for thedramatic improvement of doxorubicin cytostatic activity, when deliveredby peptide-targeted pH-sensitive liposomes.

TABLE 1 Cytotoxicity of several formulations of DXR against MDA-MB-435Sor HMEC-1 cell lines. SL[DXR] SLF3[DXR] DXR SL[DXR] SLF3[DXR]pH-sensitive pH-sensitive Time (h) IC₅₀ (μM) ± SD HMEC-1 1 0.434 ± 0.030614.6 ± 0.029 >300 87.66 ± 0.062 47.69 ± 0.064 3 0.401 ± 0.059 89.16 ±0.108 11.54 ± 7.540 12.07 ± 2.541 6.688 ± 0.119 24 0.072 ± 0.010  31.6 ±0.661  7.2 ± 0.093  3.57 ± 0.200 0.195 ± 0.049 48 0.058 ± 0.008 3.575 ±0.052 0.856 ± 0.091  0.03 ± 0.026 ND MDA-MB-435S 1 1.608 ±0.460 >800 >300 >500 >600 3 1.449 ± 0.270 >700 230.5 ± 0.042 >400 159.0± 4.950 24 0.232 ± 0.045 >700 41.24 ± 0.069 87.33 ± 0.139 3.953 ± 0.26348 0.113 ± 0.078 >700  25.5 ± 0.049  55.2 ± 0.049 3.366 ± 0.124 Cellswere incubated with free DXR or DXR encapsulated in non-targeted orF3-targeted, pH-sensitive or non-pH-sensitive, PEG-grafted liposomes for1, 3, 24 and 48 h at 37° C. in a 95% humidity and 5% CO2 atmosphere.Afterwards, cells were washed with PBS and incubated with fresh culturemedium for a total time of 96 h. IC₅₀ was determined by the MTTproliferation assay. Data are means ± SD of three independentexperiments, each done in triplicate.

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1. A ligand-targeted delivery system comprising a targeting ligandlinked to a support carrying an agent, wherein said targeting ligandbinds nucleolin, wherein said support is a pH sensitive liposome,wherein the agent is a therapeutic, diagnostic and/or imaging agent,encapsulated, entrapped or intercalated in the support, and wherein saidliposome is capable of the pH dependent intracellular release of saidagent.
 2. The delivery system of claim 1, suitable for intravenousadministration.
 3. A method of targeting a molecule overexpressed ontumor cells and/or tumor blood vessels, comprising interaction of atarget subject with the system of claim
 1. 4. The delivery system ofclaim 1 wherein the ligand selectively binds nucleolin.
 5. Aligand-targeted delivery system comprising at least one ligand linked toa support encapsulating an agent, wherein each of the at least oneligands is a peptide comprising the amino acid sequence of SEQ ID NO:1,and wherein each of the at least one ligands specifically bindsnucleolin.
 6. The delivery system of claim 1 wherein spacer ispositioned between the ligand and the support such that the interactionof the ligand with the target is not hindered.
 7. The delivery system ofclaim 6 wherein the spacer comprises a reactive group that is also ameans of linking the ligand to the support and wherein the spaceroptionally comprises a tag to facilitate recovery or identification ofthe system.
 8. The delivery system of claim 1, wherein the support is aliposome, micelle, lipid micelle, microsphere, nanosphere, chamberedmicrodevice, emulsion, lipid disc, polymer, cell, viral particle orvirus.
 9. The delivery system of claim 1, wherein the support is aliposome comprising fully hydrogenated soy phosphatidylcholine,methoxy-polyethylene glycol phosphatidylethanolamine,maleimide-polyethylene glycol phosphatidylethanolamine,N-methylpalmitoyloleoylphosphatidylcholine, phosphatidylserine,phosphatidylcholine, palmitoyloleoylphosphatidylcholine,dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine,phosphatidylglycerol, dioleoylphosphatidylethanolamine, cholesterylhemisuccinate, cholesterol, or a combination thereof.
 10. The deliverysystem of claim 1 wherein the agent is a cytotoxic, an anti-cancer,anti-inflammatory, an anti-angiogenic, an angiolytic, a vasculardisrupting agent or a photodynamic therapeutic agent.
 11. The deliverysystem of claim 1 wherein the agent is encapsulated, entrapped, orintercalated within the core of the support.
 12. The delivery system ofclaim 11, wherein the agent is one or more selected from the groupconsisting of alkylating drugs; cytotoxic antibiotics; antimetabolitei;vinca alkaloids; amsacrine; altetarmine; crisantaspase; dacarbazine;temozolomide; hydroxycarbamide (hydroxyurea); pentostatin; platinumcompounds; porfimer sodium; procarbazine; razoxane; taxanes;topoisomerase I inhibitors; trastuzumab; tretinoin; SN-38; ET-743; TLK286; anti-inflammatory agents; antiangiogenic agents or angiolyticagents; ABT-627; Bay 12-9566; Benefin; Bevacizumab; BMS-275291;cartilagederived inhibitor(CDI); CAI; CD59 complement fragment;CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagenXVIIIfragment); Fibronectin fragment; Grobeta; Halofuginone; Heparinases;Heparin hexasaccharide fragment; HMV833; Human chorionicgonadotropin(hCG); IM-862; Interferonalpha/beta/gamma; Interferon inducible protein(IP-10); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat;Metalloproteinase inhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS27023A); MoAbIMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placentalribonuclease inhibitor; Plasminogen activator inhibitor; Plateletfactor-4 (PF4); Prinomastat; Prolactin16 kD fragment; Proliferin-relatedprotein (PRP); PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS3304; SU 5416; 5U6668; SU11248; Tetrahydrocortisol-S;tetrathiomolybdate; thalidomide; Thrombospondin-1(TSP-1); TNP-470;Transforming growth factor-beta (TGF-b); Vasculostatin; Vasostatin(calreticulin fragment); ZD6126; ZD 6474; farnesyl transferaseinhibitors (FTI); bisphosphonates; and porphyrins.
 13. A method oftreating cancer in a subject by the inhibition of tumor growth and tumorblood vessel development, comprising administering to the subject aneffective amount of the delivery system of claim
 1. 14. The method ofclaim 13, wherein the delivery system is capable of providing, at least,a two-fold increase in the amount delivered of therapeutic agent to thetarget cells upon triggered-released, as compared to other deliverysystems.
 15. The delivery system of claim 1, wherein intracellulartriggered release of the agent is a function of the pH value of thetarget microenvironment.
 16. The delivery system of claim 1 wherein theagent is released through the support destabilization in acidicenvironment.
 17. The delivery system of claim 16 wherein the acidic pHenvironment comprises the endosome compartment of cells.
 18. Thedelivery system of claim 1 labelled with a radionuclide or a fluorescentmolecule.
 19. A method of identifying the presence of a target moleculeon tumor and/or endothelial cells of tumor blood vessels comprisingcontacting the tumor and/or endothelial cells of tumor blood vesselswith the system described in claim
 18. 20. A method of imaging tumorsand/or tumor vasculature in a subject comprising administering aneffective amount of the delivery system of claim
 19. 21. The deliverysystem of claim 13, wherein the alkylating drugs are one or more ofcyclophosphamide, chlorambucil, melphalan, busulfan, lomustine,carmustine, chlormethine (mustine), estramustine, treosulfan, thiotepa,or mitobronitol.
 22. The delivery system of claim 13, wherein thecytotoxic antibiotics are one or more of doxorubicin, epirubicin,aclarubicin, idarubicin, daunorubicin, mitoxantrone (mitozantrone),bleomycin, dactinomycin or mitomycin.
 23. The delivery system of claim13, wherein the antimetabolites are one or more of methotrexate,capecitabine, cytarabine, fludarabine, cladribine, gemcitabine,fluorouracil, raltitrexed (tomudex), mercaptopurine, tegafur ortioguaninc.
 24. The delivery system of claim 13, wherein the vincaalkaloids are one or more of vinblastine, vincristine, vindesine,vinorelbine or etoposide.
 25. The delivery system of claim 13, whereinthe platinum compounds are one or more of carboplatin, cisplatin oroxaliplatin.
 26. The delivery system of claim 13, wherein the taxanesare one or more of dOcetaxel or paclitaxel.
 27. The delivery system ofclaim 13, wherein the topoisomerase I inhibitors are one or both ofinotecan or topotecan.
 28. The delivery system of claim 13, wherein theanti-inflammatory agents are one or more of ibuprofen, aceclofenac,acemetacin, azapropazone, celecoxib, dexketoprofen, diclofenac sodium,diflunisal, cetodolac, fenbufen, fenoprofen, flubiprofen, indomethacin,acetaminocin, piroxicam, rofecoxib, sulindac, tenoxicam, tiaprofenuicacid, aspirin or benorilate.
 29. The delivery system of claim 13,wherein the antiangiogenic agents or angiolytic agents are one or moreof Angiostatin (plasminogen fragment), antiangiogenic antithrombin IIIor Angiozyme.